Abstract
This report describes the complete genome sequence of a double-stranded RNA (dsRNA) virus infecting the oomycetous plant pathogen Phytophthora cactorum. The virus genome consists of a single dsRNA segment of 5699 bp with two open reading frames predicted to overlap with each other and encoding a putative capsid protein of 705 aa and an RNA-dependent RNA polymerase of 779 aa. Sequence comparisons indicated that this virus, designated as “Phytophthora cactorum RNA virus 1” (PcRV1), shares the highest sequence similarity with the unclassified Pythium splendens RNA virus 1 (58% RdRp aa sequence identity). Phylogenetic analysis revealed that these two oomycete viruses group together with Giardia lamblia virus (GVL; family Totiviridae) and several unclassified toti-like viruses from arthropods, fish and fungi. This is the first report of a toti-like virus in a member of the genus Phytophthora and the first virus characterized in P. cactorum.
Highlights
Phytophthora cactorum (Leb. and Cohn) Schröeter is an oomycetous phytopathogen of the Kingdom Stramenopila that causes root, collar and crown rot as well as foliar and fruit infections on a broad range of hosts, including over 200 species of trees, ornamentals, and fruit crops [5]
No viruses had been described in P. cactorum, whereas a few viruses are known in congeneric host species: alphaendornaviruses infect members of the Phytophthora taxon ‘douglasfir’ and P. ramorum [9], and four double-stranded RNA (dsRNA) viruses have been described in the potato late blight pathogen P. infestans [2]
We report the complete genome sequence of a new toti-like virus infecting P. cactorum, designated as "Phytophthora cactorum RNA virus 1" (PcRV1)
Summary
Phytophthora cactorum isolate BirchT_KT09 was isolated in 2009 from a trunk lesion on silver birch (Betula pendula Roth.) in Denmark by Kirsten Thinggaard, cultivated on 2% malt extract agar plates, and identified based on its morphology and ribosomal internal transcribed spacer 2 (ITS 2) sequence. A 5.7-kb dsRNA element was purified from an agarose gel (Fig. 1A), and cDNA synthesis was conducted using tagged random hexamer priming followed by PCR amplification as described by Márquez et al [11] except that Maxima H- Reverse Transcriptase and DyNAzyme II DNA polymerase (Thermo Scientific) were used. The whole sequence was determined based on direct Sanger sequencing of overlapping PCR amplicons (< 1000 bp with DreamTaq DNA polymerase and > 1000 bp with Phusion High-Fidelity DNA Polymerase, Thermo Scientific), sequencing the products of at least two separate PCR reactions to cover each nucleotide position. The terminal sequences were determined using T4 RNA adapter ligation followed by PCR amplification with specific primers and Phusion High-Fidelity DNA Polymerase (Thermo Scientific). Phylogenetic analysis was carried out using the MrBayes program implemented in Geneious R10 with the following parameters: rate matrix LG + G + I with five gamma categories; 1.1 × 106 cycles for the MCMC algorithm, sampling one tree per 200 cycles; discarding 105 samples as burn-in
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